The present invention relates to the pharmaceutical use of a substance which is an interleukin 10 (IL-10) agonist, in particular the use of a substance of the invention for the manufacture of a pharmaceutical composition for prevention and/or treatment of diseases, the pathogenesis of which is related to the decreased production and/or function of immunoinhibitory mediators, especially cytokines, and/or is related to an increased production and/or function of certain immuno-inflammatory mediators, especially cytokines. In particular, the invention relates to the use of a substance of the invention for the manufacture of a pharmaceutical composition for prevention and/or treatment of auto-immune diseases (diabetes mellitus, type I; inflammatory diseases of the gastro-intestinal tract; rheumatoid arthritis), arthritis urica (gout), allergy of the skin; allergic reactions in the skin, lungs and respiratory tract (including asthma bronchiale); tissue damage as a result of hypoxia/ischemia (infarction; reperfusion); atherosclerosis; psoriasis; granulomatous disease; chronic myeloid leukaemia; acute myeloid leukaemia; cancer; graft vs. host reaction and conditions related to transplant rejection; fibrosis of the lung; fibrosis of the liver; chronic non-infectious inflammation of the lung; glomerulonephritis; pre-term labour; periodontitis; inflammatory reactions due to virus infections, osteoporosis, septic shock and/or for the manufacture of an anti-conceptive agent.
Research from the last two decades has shown that the initiation, regulation and ending of inflammatory reactions as well as the regulation of growth and differentiation within the mammalian organisms is under tight control by a special group of signal polypeptides generally called cytokines. Cytokines are polypeptides which can be produced by most nucleated cells and which transmit regulatory signals between cells, thus forming a communication network between identical or different cell types of the organism. The cytokines are extremely potent mediators and active at concentrations down to 10xe2x88x9215M. Cytokines are also key factors for the development of cellular immune reactions, which in turn form the basis for the clinical manifestations of inflammation due to infection, allergy, trauma, graft vs. host reactions and auto-immune diseases. The allergic and auto-immune diseases are explained by abnormalities in the immune system, especially in the T lymphocyte-mediated immunity, but generally these diseases are of unknown etiology. In vitro studies, animal experiments and clinical experimental studies have shown that cytokines play important pathophysiological roles for the inflammatory reactions related to auto-immune diseases, allergy, ischemia, reperfusion injury, trauma, infections, and are important for the development of cancer, atherosclerosis, pregnancy and fetal development, bone homeostasis. Cytokines may be involved in other immunoinflammatory and proliferative diseases as will be described in further detail in the following.
The said diseases are usually chronic and the treatment is palliative, i.e. most of the drugs prescribed in connection with the said diseases are directed at the allaying of symptoms and usually have no curative effect. Other treatments are so-called substitution therapies which involve life-long supplying to the patient of substances, e.g. hormones, needed due to a reduced/insufficient internal production of said substance. Said treatments are often unsatisfactory, imply unwanted and often serious side-effects, and merely delay rather than prevent the progression of the disease. Thus, improved methods of treatments and improved pharmaceutical compositions are highly needed.
Interleukin-10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine, identified in both the murine and human organism. Murine interleukin 10 (mIL-10) was originally described as a cytokine synthesis inhibitory factor released from TH2 helper T-cell clones, but it also carries proliferative effects upon various subsets of lymphocytes, including an enhancing effect upon cloning efficacy of CD4xe2x88x92,8+ murine splenic T cells (4). Human interleukin 10 (hIL-10) has recently been sequenced and revealed to have high homology with mIL-10 at DNA sequence level as well as on amino acid level. Furthermore, swine interleukin 10 has recently been sequenced and revealed to have high homology with human IL-10 at DNA sequence level as well as on amino acid level (88), see also FIG. 2. Also, hIL-10 has high homology with an open reading frame in the Epstein-Barr virus genome, BCRF1, and viral IL-10 does show some activity similar to hIL-10, cf. FIG. 1 (5).
Human IL-10 is produced by activated T cell clones and immortalized B cells, and in addition to its cytokine synthesis inhibitory factor (CSIF) activity, inhibiting the production of several pro-inflammatory cytokines and colony-stimulating factors, it also induces the production of a natural interleukin-1 receptor antagonist protein/peptide (IRAP) by mononuclear cells, thereby indirectly inhibiting IL-1 activity. IL-10 also downregulates its own production by monocytes and inhibits the expression of class II MHC expression (12). Further, hIL-10 reduces antigen-specific proliferation of human T cells and CD4+ T cell clones, when using monocytes as antigen-presenting cells. In vivo experiments in mice indicate that the outcome of Leishmania infection is dependent upon the cytokine profile from responding CD4+ T lymphocytes (13). In C57BL/6 mice resistant to Leishmania infection, CD4+ T cells from draining lymph nodes show up-regulation of IFN-xcex3 and IL-2 cytokines, whereas the sensitive BALB/c mice in their draining lymph nodes have CD4+ responding T cells releasing IL-4 and IL-10, which could be demonstrated to correlate with disease progression (13). Thus, IL-10 may exert potent regulatory effects on immunological responses both in vitro and in vivo. Further IL-10 strongly affects chemokine biology since human interleukin 10 is a specific chemotactic factor towards CD8+T cells, while IL-10 suppresses the ability of CD4+, but not CD8+, T cells to migrate in response to the T cell chemotactic cytokine, IL-8 (14). IL-10 also inhibits the chemotactic effect of other chemokines MCP-1/MCAF and RANTES (75). Since IL-10 is a deactivator of monocyte/macrophage functions and an inhibitor of Th1 activity, drugs with full or partial IL-10-like activity may possess therapeutic effect in diseases characterized by imbalance in cytokine production and/or activities.
It has previously been proposed to prepare pharmaceutical compositions comprising hIL-10 or vIL-10, and the use of hIL-10 or vIL-10 for the manufacture of a pharmaceutical composition for the treatment of various conditions such as septic or toxic shock, rheumatoid arthritis, graft-vs-host disease, tissue rejection, diabetes mellitus, autoimmune disorders, leukaemia and cancer has been disclosed in e.g. WO93/02693 and WO94/04180. Moreover, IL-10 antagonists, e.g. antibodies specifically binding to IL-10, have been disclosed in e.g. EP 405 980 and WO94/06473, and it has been contemplated that such antibodies could be useful in the treatment of HIV infected patients.
According to the present invention, it has been found that a substance other than human interleukin 10 which has one or more of the following properties:
a) induces inhibition of spontaneous IL-8 production by human monocytes,
b) induces inhibition of IL-1xcex2 induced IL-8 production by human peripheral blood mononuclear cells (PBMC),
c) induces production of interleukin-1 receptor antagonistic protein (IRAP) by human monocytes,
d) induces chemotactic migration of CD8+ human T lymphocytes in vitro,
e) desensitizes human CD8+ T cells resulting in an unresponsiveness towards rhIL-10,
f) suppresses the chemotactic response of CD4+ human T lymphocytes towards IL-8,
g) suppresses the chemotactic response of human monocytes towards MCAF/MCP-1,
h) does not inhibit class II MHC molecule expression on human monocytes, in contrast to human IL-10,
i) induces the production of IL-4 by cultured normal human CD4+ T cells,
j) reduces the TNFxcex1 production in human mixed leukocyte reaction,
such as a polypeptide which comprises the amino acid sequence Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn (SEQ ID NO:1) or an analogue or variant of said sequence (a nona-peptide with sequence homology to hIL-10, called IT9302), and derivatives thereof can be used for the prevention and/or treatment of certain forms of inflammatory processes, especially forms related to the immune and/or hormone system. It is contemplated (as described in detail in the following description of immunological mechanisms) that the action mechanism is via interference with the action of mediators of the immune system, in particular cytokines such as monokines, lymphokines, chemokines and monokine-receptor antagonists, i.e. that the substance of the invention interferes with/suppresses the production and/or action of certain cytokines and thus inhibits pathological processes leading to tissue damage, and that the substance of the invention induces the production of natural monokine-receptor antagonists thus interfering with/suppressing the action of certain cytokines and thereby inhibiting pathological processes which lead to tissue damage.
An important embodiment of the present invention thus relates to a pharmaceutical composition comprising, as the active ingredient, a substance of the invention. Other embodiments of the invention are a substance which is capable of neutralizing one or more of the activities a) to g) mentioned above, e.g. an antibody, and a pharmaceutical composition comprising such a substance.
In a further aspect, the present invention relates to the use of a substance of the invention for the manufacture of a pharmaceutical composition for substantially inhibiting a biological effect related to a cytokine, i.e. the use of a substance of the invention as an IL-1 receptor antagonist protein/peptide, lymphokine, monokine, interleukin, interferon, chemokine or colony-stimulating factor. Another aspect relates to the use of a substance of the invention for the manufacture of a pharmaceutical composition for the prophylaxis or treatment of a condition related to the disturbance of a cytokine system, i.e. the IL-1 receptor antagonist protein/-peptide, lymphokine, monokine, interleukin, interferon, chemokine or colony-stimulating factor system. In another aspect, the invention also relates to a method of treating a condition in a human related to a disturbance in a cytokine system which method comprises administering to the subject an effective amount of a substance of the invention.
The cellular immune system takes part in the development of such disorders as infectious, inflammatory and neoplastic diseases. Immunocompetent cells and their products may play important roles in the initiation, progression and possible chronic nature of development of inflammatory conditions. These disorders are often without known etiology and includes common diseases such as diabetes mellitus, rheumatoid arthritis, inflammatory diseases of the gastro-intestinal tract and of the skin. Apart from these examples, cell-mediated immunity or pro-inflammatory mediators, however, contribute to many other inflammatory and proliferative diseases (see Table 2).
Cytokines:
T-lymphocytes orchestrate the induction and regulation of cell-mediated immune reactions, and cytokine products (lymphokines) of the T cells initiate and control the immune response (1,2). The lymphocyte-activating mediators (lymphokines) produced by antigen-presenting cells belong to a group of polypeptides called cytokines. Cytokines are transmitters of cell-to-cell communication in both physiological and pathophysiological conditions, and may also function as hormones providing signals between the immune system and other tissues and organs. Cytokines may also be produced by cells outside the immune system and it is generally believed that all nucleated cells are capable of producing one or several cytokines. Thus, e.g. keratinocytes and fibroblasts are potent producers of cytokines and in this system cytokines may function as autocrine or paracrine hormones independent of the immune system (3).
Interleukin-10:
Murine interleukin 10 (mIL-10) was originally described as a cytokine synthesis inhibitory factor (CSIF) released from TH2 helper T-cell clones but it has also proliferative effects upon various subsets of lymphocytes, including an enhancing effect upon cloning efficacy of CD4-,8+ murine splenic T cells (4). Human interleukin 10 (hIL-10) has recently been described (5) and has high homology with an open reading frame in the Epstein-Barr virus genome, BCRF1, and viral IL-10 does show some activity similar to hIL-10. In the following, the biochemical, biological, physiological and possible pathophysiological roles of IL-10 will be summarized.
IL-10 Structure:
The primary structures of mouse (mIL-10) and human IL-10 (hIL-10) revealed a high degree of nucleotide sequence homology ( greater than 80%) throughout their entire length (4, 5). The only significant difference is the insertion of a human Alu repetitive sequence element in the 3xe2x80x2-untranslated region of the hIL-10 cDNA clone. The mIL-10 and hIL-10 cDNAs encode very similar open reading frames (ORF) of 178 amino acids, including hydrophobic leader sequences and corresponding to 73% amino acid homology. mIL-10, which is active on murine cells, does not cross-react significantly on human cells. hIL-10 is an 18 kDa polypeptide which lacks detectable carbohydrate, but mIL-10 is N-glycosylated at a site near its N-terminus which is missing from hIL-10. Both mIL-10 and recombinant hIL-10 (rhIL-10) are expressed as non-covalent homodimers. The extent to which mIL-10 or hIL-10 monomers are biologically active is not yet certain. mIL-10 and hIL-10 with polypeptide xe2x80x9ctagsxe2x80x9d of at least 8 amino acids at the N-terminus and 21 amino acids at the C-terminus showed no detectable loss of activity according to a publication from Moore et al. who were the first to sequence human IL-10 (6). Tagging C and N terminal ends of the whole IL-10 does not necessarily cause functional changes, since tagging is occasional or incidental. The high number of possible amino acid substitutions as described in further detail in the following might also explain while tagging failed to show missing functions. Recombinant mIL-10 and hIL-10 have been expressed in: CDS7 cells, mouse myeloma cells, chinese hamster ovary cells, a baculovirus expression system, and E. coli. The biological activities of these rIL-10 proteins are so far indistinguishable (6).
The mIL-10 gene comprises five exons arrayed over approximately 5.1 kb of DNA. The genomic clone itself encodes an expressible mIL-10 protein. The mIL-10 and hIL-10 genes are on mouse and human chromosome 1 (6, 7).
mIL-10 and hIL-10 exhibit strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus genome, BCRF1, and the homology is confined to the mature protein coding sequence and is not detected in the signal sequence or 5xe2x80x2- and 3xe2x80x2-untranslated sequences (5). Of the three sequences, mIL-10 and hIL-10 are the more closely related pair at the DNA sequence level (81%), while the DNA sequences encoding the mature hIL-10 and BCRF1 proteins have 71% homology. The homology between hIL-10 and BCRF1 is 84% on the amino acid level. It is hypothesized that the mIL-10 and hIL-10 genes are evolved from a common ancestor, while BCRF1 represents an ancestral processed, captured cellular cytokine gene and that the BCFR1 protein has been constrained to resemble hIL-10. BCFR1 is expressed during the lytic cycle of the EBV. The BCFR1 ORF encodes a 17 kDa secreted polypeptide which, like hIL-10, contains little or no glycosylation. BCFR1 displays some activities of IL-10 and has been called viral IL-10 (vIL-10), although its activity has been found to be 10% of hIL-10.
IL-10 Activity on Cytokine Production:
hIL-10 inhibits the production of a number of cytokines including interferon-xcex3 (IFN-xcex3), Tumor Necrosis Factor-xcex1 (TNF-xcex1), Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), Granulocyte-CSF (G-CSF), IL-1xcex1, IL-1xcex2, IL-2, IL-6, IL-8 and Monocyte Chemotactic polypeptide-1 (MCP-1/MCAF) by monocytes/macrophages and/or T lymphocytes (4, 5). IL-10 also inhibits the ability of monocytes to migrate as a response to the chemokine MCP-1/MCAF (75). Further, hIL-10 induces the production of an endogenous, natural interleukin-1 receptor antagonist (IRAP) (6), which inhibits IL-1xcex1 and IL-1xcex2 by competing with receptor binding. Since IL-8 is strongly inducible by IL-1xcex1 and by IL-1xcex2, IL-10 exerts part of its inhibitory effect on IL-8 production by stimulating the production of the IL-1-receptor antagonist IRAP. This last mechanism is of considerable importance for the present invention as described and exemplified in the following. IRAP has anti-inflammatory activities (9), and its therapeutic effect in rheumatoid arthritis has been suggested (10). Also, IRAP proved to be effective in the treatment of sepsis syndrome and a dose-dependent, 28-day survival benefit was associated with IRAP treatment (p=0.015) in a study by Fisher et al. (11). IRAP may exert parts of its anti-inflammatory effects by inhibiting chemokine-production such as the production of IL-8.
IL-10 and Antigen Expression:
IL-10 inhibits the expression of class II MHC expression on human monocytes (8). Constitutive and IL-4 or IFN-xcex3 induced expression of HLA-DR/DP and DQ was inhibited by hIL-10 (12). In addition, monocytes pre-incubated with IL-10 are refractory to subsequent induction of class II MHC expression by IL-4 or IFN-xcex3. IL-10 inhibits class II expression by human monocytes following activation by LPS (12, 76). BALB/c mice given 1 to 10 mg of IL-10 concomitantly with a lethal dose of LPS were protected from death (6).
IL-10 inhibits nitrogen intermediates and superoxide anions. IL-10 also inhibits reactive nitrogen intermediate (NO) as well as reactive oxygen intermediates (H2O2) by macrophages following activation by IFN-xcex3 (13).
IL-10 and T Cell Activity:
IL-10 has also modulatory effects on T cell functions/activity. Thus, hIL-10 is a potent chemotactic factor to CD8+ T lymphocytes, while hIL-10 does not show chemotaxis towards CD4+ T cells (14). Additionally, IL-10 suppresses the capacity of CD4+ T cells to respond to the chemotactic signals of the xcex2-chemokine RANTES as well as the xcex1-chemokine IL-8. hIL-10 also directly inhibits the proliferation of human peripheral blood T cells and CD4+ T cell clones (14).
IL-10 and B Lyzrphocytes:
hIL-10 co-stimulates B-lymphocyte proliferation induced by cross-linking surface Ig with immobilized anti-IgM antibody, and this effect is enhanced when B-cells are stimulated by cross-linking of their CD40 antigen with anti-CD40 antibody and mouse L cells expressing human Fcxcex3RII/CD32 (15). The effect of IL-10 on proliferation and differentiation of activated human B cells suggests that this cytokine may account for much of the superior ability of T cells expressing hIL-10 to provide help for B cell responses.
IL-10 as a Homeostatic Factor for the Immune System:
The physiological consequences of the functions of IL-10, mentioned above, are believed to be a certain degree of homeostasis on the immune system. Thus, IL-10 clearly inhibits helper T cells functions and probably stimulates T cells with suppressor functions. Therefore, like IL-4, IL-10 is believed to regulate the balance between Th1 and Th2 cytokine profiles of T cells. Especially, it is believed that IL-10 inhibits the differentiation of Th1 cells. Since Th1 cells are characterized by the production of cytokines (IFN-xcex3 and IL-2) which favour cell-mediated immune responses while Th2 cells produce cytokines (IL-4, IL-5 and IL-10) which favour a humoral response and suppress a cell-mediated immune response, IL-10 is likely to suppress a T cell mediated immune reaction such as delayed type hypersensitivity reactions, while it favours humoral responses.
As a consequence of the features of IL-10 mentioned above, the activity of IL-10 has been described as an inhibitor of macrophage and Th1 cytokine synthesis. Therefore it has been studied whether lack of IL-10 production and/or activity may play a role in diseases where an enhanced cell-mediated immunoreactivity is believed to play a role for the disease, such as in auto-immune diseases and inflammation. Anti-IL-10 antibody-treated mice show a stronger inflammatory response to monokine-induced inflammation and are significantly more susceptible to death induced by LPS-induced septic shock, a monokine-mediated inflammatory reaction (16). Also, IL-10 knock-out mice spontaneously develop inflammatory reactions of the gut similar to that of colitis ulcerosa (17). Additionally, it has been investigated whether IL-10 plays a role in different parasite, mycobacterial or viral infections, and IL-10 has been shown to play a pathophysiological role in the immune-paresis related to the infection with Schistosoma mansoni (18). Also a role in Mycobacterium leprae infections has been suggested. Recently, it has been found that AIDS patients with a poor prognosis have a higher level of IL-10 in plasma, and it has been suggested that this contributes to the immune paresis which is known from AIDS (19).
Therapeutic Considerations:
These in vivo results/data strongly suggest a homeostatic role of IL-10 in controlling cell-mediated and monokine-amplified immune inflammation and indicate the wide-ranged therapeutical applications of IL-10 or a drug with IL-10-like activity in the treatment of diseases which are characterized by a decreased/insufficient production and/or activity of IL-10. Since the substance of the present invention exemplified by IT9302 exerts IL-10-like activity by
1) inducing IRAP production by human monocytes,
2) inhibiting spontaneous IL-8 production by human monocytes,
3) inhibiting IL-1xcex2-stimulated IL-8 production by peripheral blood mononuclear cells (PBMC),
4) stimulating chemotactic migration of CD8+, but not CD4+, human T lymphocytes,
5) desensitizing human CD8+ T cells towards rhIL-10-induced chemotactic migration,
6) inhibiting IL-8-mediated human CD4+ T cell chemotaxis, and
7) inhibiting MCP-1/MCAF-mediated human monocyte chemotaxis,
8) inducing the production of IL-4 by cultured normal human CD4+ T cells,
9) reducing the TNFxcex1 production in human mixed leucocyte reaction,
this polypeptide and analogues thereof may thus possess the same therapeutic possibilities as hIL-10. Table 3 lists some diseases where an immune-modulator like IL-10 or an immunemodulator with IL-10-like activity may have therapeutic importance:
Partial sequences of hIL-10 having a length of 9 amino acids was chosen according to the principle that the sequences should possess high homology between vIL-10 and hIL-10, but as low homology to mIL-10 as possible. This strategy was based on the fact that vIL-10 cross-reacts partly with hIL-10, while mIL-10 does not cross-react with hIL-10 (see above). Thus, sequences of the hIL-10 responsible for certain activities in the human organism may be located at domains where there is high homology between hIL-10 and vIL-10 but low in relation to mIL-10.
The signal polypeptide of hIL-10 is supposed to consist of the first 18 amino acids. The mature protein starts from amino acid No. 19 (No. 1 in the functional protein, which is a serine), and the total protein is containing 160 amino acids. By looking at the human and viral IL-10 COOH-terminal sequences and in particular position 157 and 159, containing lysine and arginine residues, this domain was found to be favourable for being partly responsible for receptor binding. (FIGS. 1 and 2).
After screening several candidates, obtained by chemical synthesis, for IL-10-like activity, it was found that a synthetic nonapeptide, IT9302, possessed some immuno-suppressive activities which mimic those of hIL-10 as described in further detail in the following examples. IT9302 corresponds to a nonapeptide sequence from the C-terminal end of hIL-10 with the following amino acid sequence:
NH2-Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn-COOHxe2x80x83xe2x80x83(SEQ ID NO:1)
IT9302 has 100% homology with a segment of hIL-10 corresponding to amino acids Nos. 152 to 160 at the C-terminal end. Furthermore, this polypeptide sequence has 6 amino acids in common with the virus vIL-10 in the corresponding area, see FIGS. 1 and 2, which means that 6 of 9 or 66% of the amino acids are homologous as defined herein, and 4 amino acids in common with mIL-10, i.e. 4 of 9 or 44%. This nonapeptide is the only sequence obtained or derived from hIL-10 and examined until now which has the special properties demonstrated in the examples. Until contemplated by the present inventors, no one has pointed to this sequence as a functional domain.
In its broadest aspect, the present invention thus relates to a substance which exhibits hIL-10 agonist activity, i.e. a substance other than human interleukin 10 which has one or more of the following properties:
a) induces inhibition of spontaneous IL-8 production by human monocytes,
b) induces inhibition of IL-1xcex2 induced IL-8 production by human peripheral blood mononuclear cells (PBMC),
c) induces production of interleukin-1 receptor antagonistic protein (IRAP) by human monocytes,
d) induces chemotactic migration of CD8+ human T lymphocytes in vitro,
e) desensitizes human CD8+ T cells resulting in an unresponsiveness towards rhIL-10,
f) suppresses the chemotactic response of CD4+ human T lymphocytes towards IL-8,
g) suppresses the chemotactic response of human monocytes towards MCAF/MCP-1,
h) does not inhibit class II MHC molecule expression on human monocytes, in contrast to human IL-10,
i) induces the production of IL-4 by cultured normal human CD4+ T cells,
j) reduces the TNFxcex1 production in human mixed leukocyte reaction.
Among these activities, d) to g) are considered the most unique. One important embodiment of the invention is a substance which exhibits hIL-10 agonist activity as defined above and which comprises the amino acid sequence Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn (SEQ ID NO:1) or an analogue or variant of said sequence. This sequence, and all other polypeptide sequences in the present specification and claims, are, also when not explicitly stated, written from the N-terminal to the C-terminal end in the conventional format.
The nonapeptide IT9302 is very potent to induce different functions and is very stable, and it is presumed that it can not be incorrectly coupled to receptors. A ronapeptide has been chosen because generally a 9 amino acid polypeptide sequence is unique for a protein. However, it is to be noted that the 6 amino acids at the very end of hIL-10 seem to be the most important ones. Within the scope of the present invention is thus a substance or-polypeptide comprising a subsequence of the amino acid sequence Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn (SEQ ID NO:1).
It is considered likely that some amino acid substitutions will not have adverse effects on the hIL-10 agonist activity as defined herein as long as the threonine, the lysine and the arginine are present and with one amino acid placed inbetween.
In one aspect, the present invention thus relates to a polypeptide having the formula
Thr-X4-Lys-X5-Arg-X6-xe2x80x83xe2x80x83(SEQ ID NO:19),
wherein
X4 and X5 are independently selected from the group consisting of Met, Ile, Leu and Val; and
X6 is selected from the group consisting of Asn, Asp, Gln and Glu, with the proviso that the polypeptide is not Thr-Met-LyB-Ile-Arg-Asn.
In another aspect, the invention relates to a polypeptide having the formula
X3-Thr-X4-Lys-X5-Arg-X6xe2x80x83xe2x80x83(SEQ ID NO:20),
wherein
X3, X4 and X5 are independently selected from the group consisting of Met, Ile, Leu and Val; and
X6 is selected from the group consisting of Asn, Asp, Gln and Glu.
In yet a further aspect, the invention relates to polypeptides having the formula
X2xe2x80x94X3-Thr-X4-Lys-X5-Arg-X6xe2x80x83xe2x80x83(SEQ ID NO:21),
wherein
X2 is Tyr or Phe,
X3, X4 and X5 are independently selected from the group consisting of Met, Ile, Leu and Val; and
X6 is selected from the group consisting of Asn, Asp, Gln and Glu.
Preferred embodiments of the invention are polypeptides having the formula
X1xe2x80x94X2xe2x80x94X3-Thr-X4-Lys-X5-Arg-X6xe2x80x83xe2x80x83(SEQ ID NO:22),
wherein
X1 is Ala or Gly,
X2 is Tyr or Phe,
X3, X4 and X5 are independently selected from the group consisting of Met, Ile, Leu, and Val; and
X6 is selected from the group consisting of Asn, Asp, Gln and Glu.
The present invention thus relates to a substance or polypeptide which comprises an amino acid sequence as defined above, with the proviso that the substance or polypeptide is not human IL-10.
Examples of specific polypeptides which are presumed to have hIL-10 agonist activity as defined above are as follows:
1. NH2-Ala-Tyr-Met-Thr-Ile-Lys-Met-Arg-Asn-COOH (SEQ ID NO:2)
2. NH2-Ala-Phe-Met-Thr-Leu-Lys-Leu-Arg-Asn-COOH (SEQ ID NO:3)
3. NH2-Ala-Tyr-Met-Thr-Met-Lys-Val-Arg-Glu-COOH (SEQ ID NO:4)
4. NH2-Gly-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asp-COOH (SEQ ID NO:5)
5. NH2-Ala-Phe-Met-Thr-Met-Lys-Ile-Arg-Asp-COOH (SEQ ID NO:6)
6. NH2-Ala-Tyr-Ile-Thr-Met-Lys-Ile-Arg-Asp-COOH (SEQ ID NO:7)
7. NH2-Ala-Tyr-Leu-Thr-Met-Lys-Ile-Arg-Asp-COOH (SEQ ID NO:8)
8. NH2-Ala-Tyr-Val-Thr-Met-Lys-Ile-Arg-Asp-COOH (SEQ ID NO:9)
9. NH2-Ala-Tyr-Met-Thr-Ile-Lys-Ile-Arg-Asp-COOH (SEQ ID NO:10)
10. NH2-Ala-Tyr-Met-Thr-Leu-Lys-Ile-Arg-Asp-COOH (SEQ ID NO:11)
11. NH2-Ala-Tyr-Met-Thr-Val-Lys-Ile-Arg-Asp-COOH (SEQ ID NO:12)
12. NH2-Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asp-COOH (SEQ ID NO:13)
13. NH2-Ala-Tyr-Met-Thr-Met-Lys-Met-Arg-Asp-COOH (SEQ ID NO:14)
14. NH2-Ala-Tyr-Met-Thr-Met-Lys-Val-Arg-Asp-COOH (SEQ ID NO:15)
15. NH2-Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Gln-COOH (SEQ ID NO:16)
16. NH2-Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Glu-COOH (SEQ ID NO:17)
For comparison, another nonapeptide called IT9301 with 100% homology to a sequence of hIL-10 was synthesized. This polypeptide, IT9301, corresponds to a nonapeptide sequence starting at the N-terminal end of hIL-10, (amino acids nos. 19 to 27, i.e. the first 9 amino acids of the mature protein) and has the following amino acid sequence:
NH2-Ser-Pro-Gly-Gln-Gly-Thr-Gln-Ser-Glu-COOHxe2x80x83xe2x80x83(SEQ ID NO:18)
This polypeptide showed no IL-10-like activity using the techniques described below and was only chosen because it is at the other end of hIL-10 and mostly served as a control for negative results. It does not have the characteristics of the C-terminal sequence with regard to sequence homology to vIL-10, and favourable amino acids for receptor binding. IT9301 was tested in the IL-1xcex2 induced peripheral blood mononuclear cell system for IL-8 production, but did not induce any inhibition at all of the IL-8 production from the cells.
In accordance with the present invention, the term xe2x80x9ca hIL-10 agonist substance of the inventionxe2x80x9d comprises any pharmaceutically active and acceptable compound being identical or structurally similar to IT9302 and exhibiting relevant biological actions similar to those of IT9302, including derivatives of IT9302, especially pharmaceutically acceptable salts, esters and solvates as well as conjugates of IT9302 or of the IT9302 derivatives including peptido-mimetics. Covalent coupling of IT9302 at the NH2-terminal end to appropriate carriers, e.g. polyethylene glycol or sugar might extend the half-life of the polypeptide in vivo.
The following definitions are employed in the present text:
xe2x80x9cCytokinexe2x80x9d is a general term for a proteinaceous mediator released primarily but not exclusively by a cell population of the immune system as a response to a specific stimulating agent, e.g. a specific antigen or an alloantigen; or a non-specific, polyclonal activator, e.g. an endotoxin or other cell wall components of a gram-negative bacterium.
xe2x80x9cLymphokinexe2x80x9d is a general term for a proteinaceous mediator released by sensitized lymphocytes as a response to a stimulating agent, e.g. a specific antigen or an alloantigen; or by a lymphocyte challenged by a polyclonal activator, e.g. an endotoxin or other cell wall components of a gram-negative bacterium.
xe2x80x9cInterleukinxe2x80x9d is a general term for a proteinaceous mediator released primarily but not exclusively by a macrophage, T, B, or NK cell as a response to a stimulating agent, e.g. a specific antigen or an alloantigen; or by a lymphocyte challenged by a polyclonal activator, e.g. an endotoxin or other cell wall components of a gram-negative bacterium.
xe2x80x9cMonokinexe2x80x9d is a general term for a proteinaceous mediator released primarily but not exclusively by a mononuclear phagocyte (e.g. a monocyte or a macrophage or a Kupffer cell (liver) or a Langerhans"" cell (skin) as a response to any stimulating agent.
xe2x80x9cChemokinexe2x80x9d is a general term for a proteinaceous chemotactic and/or leukocyte-activating mediator released primarily but not exclusively by a cell population of the immune system as a response to a specific stimulating agent, e.g. a specific antigen or an alloantigen; or a non-specific, polyclonal activator, e.g. an endotoxin or other cell wall components of a gram-negative bacterium and belonging to a particular gene family either the chemokine-xcex1 gene family or the chemokine-xcex2 gene family.
xe2x80x9cInterferonxe2x80x9d is a general term for a proteinaceous antiviral and/or monocyte-activating mediator released primarily but not exclusively by a cell population of the immune system as a response to a virus or an interferon inducer such as a polynucleotide; in particular cells of the immune system as a response to a specific stimulating agent, e.g. a specific antigen or an alloantigen; or a non-specific, polyclonal activator, e.g. an endotoxin or other cell wall components of a gram-negative bacterium.
xe2x80x9cColony-stimulating factorxe2x80x9d is a general term for a proteinaceous, haematopoietic colony-stimulating mediator released primarily but not exclusively by a cell population of the immune system as a response to a specific stimulating agent, e.g. a specific antigen or an alloantigen; or a non-specific, polyclonal activator, e.g. an endotoxin or other cell wall components of a gram-negative bacterium.
xe2x80x9cPolypeptidexe2x80x9d as used herein means both short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues), and longer peptides (the usual interpretation of xe2x80x9cpolypeptidexe2x80x9d, i.e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, oligopeptide, or polypeptide which may be chemically modified by being glycosylated, by being lipidated, or by comprising prosthetic groups). The definition of polypeptides also comprises native forms of peptides/proteins in humans as well as recombinant proteins or peptides in any type of expression vectors transforming any kind of host, and also chemically synthesized peptides.
One embodiment of the present invention relates to a polypeptide comprising a subsequence of the sequence of human IL-10 and which has a degree of homology to vIL-10 which is 66% and a degree of homology to mIL-10 which is 44%, in particular SEQ ID NO:1. By the term xe2x80x9chomologyxe2x80x9d is meant the identity in sequences of amino acids in segments of two or more amino acids when matched with respect to identity and position of the amino acids of the polypeptides.
Homology as the term is used herein is thus a measure of similarity between two amino acid (or nucleotide) sequences. Homology is expressed as the fraction or percentage of matching amino acids (or bases) after two sequences (possibly of unequal length) have been aligned. The term alignment is used in the sense defined in (76). Roughly, two sequences are aligned by maximizing the number of matching bases (or amino acids) between the two sequences with the insertion of a minimal number of xe2x80x9cblankxe2x80x9d or xe2x80x9cnullxe2x80x9d bases into either sequence to bring about the maximum overlap.
The term xe2x80x9chomologousxe2x80x9d is thus used here inter alia to illustrate the degree of identity between the amino acid sequence of a given polypeptide and the amino acid sequence of IT9302. The amino acid sequence to be compared with the amino acid sequence of IT9302 may be deduced from a nucleotide sequence such as a DNA or RNA sequence, e.g. obtained by hybridization as defined in the following, or may be obtained by conventional amino acid sequencing methods. The degree of homology is preferably determined on the amino acid sequence of a mature polypeptide, i.e. without taking any leader sequence into consideration. Generally, only coding regions are used when comparing nucleotide sequences in order to determine their internal homology.
Although it is contemplated that a substantial degree of homology to hIL-10 and vIL-10 is beneficial, it is not unlikely that subsequences of the subsequence of hIL-10 which show a lower degree of homology to vIL-10, say, e.g., 50%, 55% or 60%, may also show one or more beneficial hIL-10 agonist activities. Moreover, as discussed above, it is not deemed absolutely necessary that the degree of homology to hIL-10 is 100%. Within the scope of the present invention are also polypeptides having a lower degree of homology to hIL-10, such as 75%, 80%, 85%, 90% or 95%, although 100% is preferred.
Such polypeptides may be considered analogues of the nonapeptide. By the term xe2x80x9can analogue or variant thereofxe2x80x9d is thus meant a polypeptide not having exactly the amino acid sequence Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn (SEQ ID NO:1), but still having xe2x80x9chIL-10 agonist activityxe2x80x9d as defined above. Generally, such polypeptides will be polypeptides which vary e.g. to a certain extent in the amino acid composition, or the post-translational modifications e.g. glycosylation or phosphorylation, as compared to IT9302 described in the examples.
The term xe2x80x9canaloguexe2x80x9d or xe2x80x9cvariantxe2x80x9d is thus used in the present context to indicate a protein or polypeptide having a somewhat different, but still similar, amino acid sequence to the amino acid sequence of IT9302, allowing for minor variations that alter the amino acid sequence, e.g. deletions, site directed mutations, insertions of extra amino acids, or combinations thereof, to generate polypeptide analogues of IT9302.
Site-directed mutagenesis provides one convenient way to effect conservative amino acid substitutions, and the like, in the native protein sequence. Alternatively, analogues may be prepared by the well known methods of liquid or solid phase polypeptide synthesis utilizing the successive coupling of the individual amino acids of the polypeptide sequence, or the polypeptide can be synthesized by the coupling of individual amino acids forming fragments of the polypeptide sequence which are later coupled so as to result in the desired polypeptide.
xe2x80x9cConservativexe2x80x9d as used herein means (i) that the alterations are as conformationally neutral as possible, that is, designed to produce minimal changes in the tertiary structure of the mutant polypeptides as compared to the native protein, and (ii) that the alterations are as antigenically neutral as possible, that is designed to produce minimal changes in the antigenic determinants of the mutant polypeptides as compared to the native protein. Conformational neutrality is desirable for preserving biological activity, and antigenic neutrality is desirable for avoiding the triggering of immunogenic responses in patients or animals treated with the substances of the invention. Although it is difficult to select with absolute certainty which alternatives will be conformationally and antigenically neutral, rules exist which can guide those skilled in the art to make alterations that have high probabilities of being conformationally and antigenically neutral, see e.g. (77) and (78). Some of the more important rules include (1) replacement of hydrophobic residues is less likely to produce changes in antigenicity because they are likely to be located in the protein""s interior, e.g. Berzofsky (cited above) and Bowie et al. (cited above); (2) replacement of physicochemically similar, i.e. synonymous, residues is less likely to produce conformational changes because the replacing amino acid can play the same structural role as the replaced amino acid; and (3) alteration of evolutionarily conserved sequences is likely to produce deleterious conformational effects because evolutionary conservation suggests sequences may be functionally important. In addition to such basic rules for selecting mutein sequences, assays are available to confirm the biological activity and conformation of the engineered molecules. Biological assays for the substances of the invention are described more fully in the examples. Changes in conformation can be tested by at least two well known assays: the microcomplement fixation method, e.g. (79) and (80) used widely in evolutionary studies of the tertiary structures of proteins; and affinities to sets of conformation-specific monoclonal antibodies, egg (81).
An important embodiment of the present invention thus relates to a polypeptide in which at least one amino acid residue has been substituted with a different amino acid residue and/or in which at least one amino acid residue has been deleted or added so as to result in a polypeptide comprising an amino acid sequence being different from the amino acid sequence or a subsequence of said amino acid sequence as defined in the following, but essentially having hIL-10 agonist activity as defined above.
An interesting embodiment of the invention relates to a polypeptide which is an analogue and/or comprises at least part of the polypeptide of the invention amounting in total from 10 to 100 amino acids, e.g. at least 12 amino acids, at least 15 amino acids, at least 20 amino acids or at least 30 amino acids.
In a preferred embodiment of the invention, the substance or polypeptide is used in substantially pure form. To obtain this, purification of the polypeptide may be required. Examples of the procedures employed for the purification of polypeptides are: (i) immunoprecipitation or affinity chromatography with antibodies, (ii) affinity chromatography with a suitable ligand, (iii) other chromatography procedures such as gel filtration, ion exchange or high performance liquid chromatography or derivatives of any of the above, (iv) electrophoretic procedures like polyacrylamide gel electrophoresis, denaturating polyacrylamide gel electrophoresis, agarose gel electrophoresis and isoelectric focusing, (v) any other specific solubilization and/or purification techniques.
Within the scope of the present invention is also a nucleotide sequence encoding a polypeptide as defined above, in particular a nucleotide sequence encoding a polypeptide comprising or being the amino acid sequence SEQ ID NO:1, e.g. a nucleotide sequence comprising the sequence GCC TAC ATG ACA ATG AAG ATA CGA AAC (SEQ ID NO:23) or a nucleotide sequence: encoding a polypeptide having a subsequence of said amino acid sequence. Moreover, a modified nucleotide sequence which differs from the nucleotide sequence above in that at least one nucleotide has been deleted, substituted or modified or at least one additional nucleotide has been inserted so as to result in a nucleotide sequence which encodes a polypeptide having hIL-10 agonist activity is within the scope of the invention.
In the present specification and claims, the term xe2x80x9csubsequencexe2x80x9d designates a sequence which preferably has a size of at least 18 nucleotides, more preferably at least 21 nucleotides, and most preferably at least 24 nucleotides. In a number of embodiments of the invention, the subsequence or analogue of the nucleotide sequence of the invention will comprise at least 27 nucleotides, such as at least 30 nucleotides or at least 45 nucleotides. The polypeptide encoded by the xe2x80x9csubsequencexe2x80x9d should conform to at least one of the criteria a)-g) above and/or the nucleotide xe2x80x9csubsequencexe2x80x9d should hybridize with the nucleotide sequence comprising the sequence SEQ ID NO:23 under high stringency conditions.
The term xe2x80x9chighly stringentxe2x80x9d when used in conjunction with hybridization conditions is as defined in the art that is 5-10xc2x0 C. under the melting point Tm, cf. Sambrook et al, 1989, pages 11.45-11.49.
The term xe2x80x9c1analoguexe2x80x9d with regard to the DNA fragments of the invention includes a nucleotide sequence which encodes a polypeptide identical or substantially identical to the polypeptide encoded by the DNA referred to above. It is well known that the same amino acid may be encoded by various codons, the codon usage being related, inter alia, to the preference of the organisms in question expressing the nucleotide sequence. Thus, one or more nucleotides or codons of the DNA fragment of the invention may be exchanged by others which, when expressed, result in a polypeptide identical or substantially identical to the polypeptide encoded by the DNA fragment shown above.
Furthermore, the terms xe2x80x9cfanaloguexe2x80x9d and xe2x80x9csubsequencexe2x80x9d are intended to allow for variations in the sequence such as substitution, insertion (including introns), addition and rearrangement of one or more nucleotides, which variations do not have any substantial adverse effect on the hIL-10 agonist activity of the polypeptide encoded by the DNA fragment or a subsequence thereof. The invention thus also includes a nucleotide sequence encoding a polypeptide having a subsequence of the amino acid sequence SEQ ID NO:1.
The polypeptides of the invention can be produced using recombinant DNA technology. An important embodiment of the present invention relates to an expression system comprising a nucleotide sequence of the invention. Within the scope of the invention is thus also an expression system comprising a nucleotide sequence of the invention, such as a replicable expression vector which carries and is capable of mediating the expression of a nucleotide sequence as defined above.
The organism which is used for the production of the polypeptide of the invention may be a higher organism, e.g. an animal, or a lower organism, e.g. a microorganism such as Escherichia coli, a yeast, a protozoan, or cell derived from a multicellular organism such as a fungus, an insect cell, a plant cell, a mammalian cell or a cell line, which carries an expression system as defined above.
Irrespective of the type of organism used, the DNA fragment of the invention is introduced into the organism either directly or by means of a suitable vector. Alternatively, the polypeptides may be produced in the mammalian cell lines by introducing the DNA fragment or an analogue or a subsequence thereof of the invention either directly or by means of an expression vector. The polypeptides of the invention may also be produced by chemical synthesis as discussed above.
The invention furthermore relates to a plasmid vector containing a DNA sequence coding for a polypeptide of the invention or a fusion polypeptide as defined herein. In one particular important embodiment, the DNA fragment or an analogue or subsequence thereof of the invention or a fusion DNA fragment of the invention as defined herein may be carried by a replicable expression vector which is capable of replicating in a host organism or a cell line.
The vector may in particular be a plasmid, phage, cosmid, mini-chromosome or virus. In an interesting embodiment of the invention, the vector may be a vector which, when introduced in a host cell, is integrated in the host cell genome.
The polypeptide produced as described above may be subjected to post-translational or post-synthetic modifications as a result of thermal treatment, chemical treatment (formaldehyde, glutaraldehyde etc.) or enzyme treatment (peptidases, proteinases and protein modification enzymes). The polypeptide may be processed in a different way when produced in an organism as compared to its natural production environment. As an example, glycosylation is often achieved when the polypeptide is expressed by a cell of a higher organism such as yeast or preferably a mammal. Glycosylation is normally found in connection with amino acid residues Asn, Ser, Thr or hydroxylysine.
One embodiment of the invention thus relates to a method of producing a polypeptide as defined above comprising the following steps of:
(a) inserting a nucleotide sequence as defined above in an expression vector,
(b) transforming a suitable host organism with the vector produced in step (a),
(c) culturing the host organism produced in step (b) under suitable conditions for expressing the polypeptide,
(d) harvesting the polypeptide, and
(e) optionally subjecting the polypeptide to post-translational modification.
It is contemplated that antagonists effect to hIL-10 or vIL-10 can be achieved inter alia by using antibodies binding specifically to the nonapeptide IT9302, and that these can be used in therapy for neutralizing high concentration of IL-10.
In another aspect, the present invention thus relates to an antibody which specifically binds to the polypeptide of the invention.
The term xe2x80x9cantibodyxe2x80x9d refers to a substance which is produced by a mammal or more precisely a cell of mammalian origin belonging to the immune system as a response to exposure to a polypeptide antigen of the invention. In the present specification and claims xe2x80x9can antibodyxe2x80x9d is defined as consisting essentially of the specifically binding basic unit which consists of two heavy chains and two light chains. In its broadest aspect, however, the concept of an antibody should also include e.g. a dimer or pentamer of the basic unit.
The variant domain of an antibody is composed of variable and constant sequences. The variant part of the domain is called the idiotype of the antibody. This part of the antibody is responsible for the interaction with the antigen, the antigen binding. In the present context, the term antibody is understood as the whole antibody molecule or any fragments thereof. An antibody can be fragmented during and/or after the production. It can also be made in the fragmented form to begin with and used as such or used after joining different fragments. Especially interesting fragments are binding fragments of the antibodies of the invention, e.g. Fab or Fabxe2x80x2 fragments.
The idiotypic (antigen binding) structure of the antibody is antigenic and can thus give rise to specific antibodies directed against the idiotypic structure. The antibodies raised against the idiotype are called the anti-idiotypic antibodies. Such antibodies may mimic the structure of the original antigen and therefore may function as the original antigen. Such antibodies may be able to substitute the original antigen for a part or all of the functions, usability and properties of the original polypeptide of the invention.
The antibodies of the invention comprise polyclonal antibodies as well as monoclonal antibodies.
The antibody or fragments thereof may be of a monospecific (polyclonal) kind. The monospecific antibody may be prepared by injecting a suitable animal with a substantially pure preparation of the polypeptide of the invention. This can be followed by one or more booster injections at suitable intervals before the first bleeding. The animals are bled about 5-7 days after each immunization. Antibodies may optionally be isolated from the serum using standard antibody purification techniques.
The animal used for the preparation of antibodies binding to the polypeptide of the invention is preferably selected from the group consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, horse and guinea pigs. The cells producing the antibodies may be spleen cells or peripheral blood lymphocytes.
A monoclonal antibody or fragments thereof may be raised against an essential component of the polypeptide, i.e. an epitope. The monoclonal antibody may be produced using conventional techniques (Kxc3x6hler and Milstein, 1975) by use of a hybridoma cell line, or by clones or subclones thereof or by cells carrying genetic information from the hybridoma cell line producing said monoclonal antibody. The monoclonal antibody may be produced by fusing cells producing said monoclonal antibody with cells of a suitable cell line, and cloning the resulting hybridoma cells producing said monoclonal antibody. Alternatively, the monoclonal antibody may be produced by immortalizing an unfused cell line producing said monoclonal antibody. The monoclonal antibodies are ultimately harvested from the cell growth medium. Hybridoma cells used to make monoclonal antibody may be grown in vitro or in the body cavity of an animal. The monoclonal antibody or fragments thereof may also be made using the recombinant DNA techniques (Huse et al. 1989).
Monoclonal antibodies may also be made by immunizing the suitable animals with a unpurified preparation of the polypeptide of the invention. The resulting hybridoma clones secreting monoclonal antibodies should be screened for their ability to binding to polypeptide(s) or its analogue.
For purposes not requiring a high specificity, the antibody may be a polyclonal antibody. Polyclonal antibodies may be obtained, e.g. as described in Harboe and Ingild, see above. More specifically, when polyclonal antibodies are to be obtained, the polypeptide of the invention or an analogue thereof is, preferably after addition of a suitable adjuvant, such as Freund""s incomplete or complete adjuvant, injected into an animal. The animals are bled regularly, for instance at weekly intervals, and the blood obtained is separated into an antibody containing serum fraction, and optionally said fraction is subjected to further conventional procedures for antibody purification, and/or procedures involving use of the purified polypeptide or an analogue thereof.
The antibody may also be an anti-anti-idiotypic antibody directed against an anti-idiotypic antibody which is an antibody directed against the site of an antibody which is reactive with the epitope on the antigen. The anti-idiotypic antibody may be prepared by a similar method to that outlined above for the monoclonal or polyclonal antibody.
Within the scope of the invention is an antibody which binds to a substance or polypeptide as defined above, in particular an antibody which binds specifically to a polypeptide having the amino acid sequence Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn.
In a broad aspect, the invention thus relates to a substance which is capable of neutralizing one or more of the hIL-10activities a) to j), i.e. having hIL-10 antagonist activity, such as an antibody having these properties. A monoclonal antibody 19F1 (82) which is capable of specifically binding to IL-10 is known and can block endogenous IL-10 produced by LPS stimulated monocytes, see Table 1 (82). This antibody recognizes the natively folded IL-10, but not necessarily a whole functional domain; however, this antibody does not specifically bind IT9302.
Within the scope of the present invention is also a pharmaceutical composition comprising a such substance as well as a pharmaceutical composition comprising a substance or polypeptide of the invention.
A very important aspect of the invention relates to a pharmaceutical composition comprising a substance having hIL-10 agonist activity or hIL-10 antagonist activity as defined above and a pharmaceutically acceptable excipient. The composition may comprise e.g. purified synthesized protein or a purified recombinant polypeptide, a monoclonal or polyclonal antibody or any other substance fulfilling the criteria a)-j) or, if having hIL-10 antagonist activity, being capable of neutralizing one or more of the hIL-10 activities a) to j).
The IL-10 agonist or antagonist used in this invention may be prepared as formulations in pharmaceutically acceptable media, for example, saline, phosphate buffered saline (PBS), Ringer""s solution, dextrose/saline, Hank""s solution, and glucose. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like. Additives may also include additional active ingredients, e.g. bactericidal agents, or stabilizers. The amount administered to the patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the host, the manner of administration, and the like.
The pharmaceutical compositions are typically intended for transdermal or parenteral administration, e.g. intravenously, subcutaneously, or intramuscularly. Orally administrative forms are also desired and can be provided by modifying the composition to bypass the stomach environment. The composition can be used for prophylactic and/or therapeutic treatment. Preferably, the pharmaceutical compositions are administered intravenously. Thus, the invention provides compositions which comprise an IL-10 agonist or antagonist substance dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The IL-10 agonist or antagonist may also be administered with a second biologically active agent, such as a standard chemotherapeutic agent. Such agents include but are not limited to vincristine, daunorubicin, L-asparaginase, mitoxantrone and amsacrine.
In therapeutic applications, the pharmaceutical compositions are administered to a patient in an amount sufficient to produce the desired effect, defined as a xe2x80x9ctherapeutically effective dosexe2x80x9d. The therapeutically effective dose of a IL-10 agonist or antagonist will vary according to, for example, the particular use for which the treatment is made, the manner of administration, the health and condition of the patient, and the judgment of the prescribing physician. For example, the dose for continuous infusion will typically be in the range of about 500 ng to about 800 xcexcg per day for a 70 kg patient, preferably between about 10 xcexcg and about 300 xcexcg. The dose will typically be between 700 ng/kg/day and 16 xcexcg/kg/day.
The concentration of IL-10 agonist or antagonist in the pharmaceutical formulations can vary widely, i.e. from about 10 xcexcg to about 5 mg/ml, preferably between about 100 xcexcg and about 2 mg/ml. The concentration will usually be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of dextrose/saline solution and 2.5 mg of IL-10 agonist or antagonist.
For solid compositions, conventional non-toxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by incorporating normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, an IL-10 agonist or antagonist substance, preferably 25-75%.
For aerosol administration, the IL-10 agonist or antagonist is preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of IL-10 agonist or antagonists are 0.01-20% by weight, preferably 1-10%. The surfactant must, of course, be non-toxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arbitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycerides may be employed.
The surfactant may constitute 0.1-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. Liquified propellants are typically gases at ambient conditions, and are condensed under pressure. Among suitable liquified propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above may also be employed. In producing the aerosol, a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided polypeptide(s) and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.
To enhance the serum half-life, the IL-10 agonist or antagonist may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or other conventional techniques may be employed which provide an extended lifetime of the polypeptides. Thus, in certain embodiments, the IL-10 agonist or antagonist may be encapsulated in a liposome. A variety of methods are available for preparing liposomes, as described in, e.g., (83), (84), (85) and (86).
One important embodiment of the invention thus relates to the use of a substance for diminishing or neutralizing high concentration of hIL-10 and/or vIL-10, such as the use of a substance having hIL-10 antagonistic properties for the manufacture of a pharmaceutical composition for the treatment or prophylaxis of ovary cancer and/or AIDS (87, 19).
Within the scope of the invention is also a method of treating and/or preventing ovary cancer and/or AIDS, the method comprising administering, to a patient in need thereof, a therapeutically or prophylactically effective amount of a hIL-10 antagonist substance as well as use of a compound which has an hIL-10 antagonist activity for the manufacture of a pharmaceutical composition for treatment or prophylaxis of ovary cancer and/or AIDS.
In accordance with the present invention, it has as described above been found that IT9302 and analogues and variants thereof are useful for preventing effects of cytokines known to be pathogenetically involved in the previously described pathological conditions.
Therefore, the potentials of therapy by using the polypeptide of the invention or analogues or derivatives thereof is contemplated and should be investigated in all diseases where a therapeutic effect of hIL-10 and/or IRAP is expected (see above, Table 3).
Very important embodiments of the invention relate to use of a substance or polypeptide of the invention for the treatment or prophylaxis of one or more of the diseases mentioned in Table 3, to the use of a substance or polypeptide according to the invention for the manufacture of a pharmaceutical composition for the treatment or prophylaxis of one or more of the diseases mentioned in Table 3, as well as to a method of treating and/or preventing one or more of the diseases mentioned in Table 3, the method comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of a substance or polypeptide according to the invention.
One important aspect of the invention is thus the use of IT9302 or a functional derivative thereof for the manufacture of a pharmaceutical composition for substantially inhibiting a biological effect in a human related to a cytokine, such as a lymphokine, interleukin, monokine, chemokine, interferon, colony-stimulating factor, prostaglandin and/or leukotriene, for the prophylaxis or treatment of a condition related to a disturbance of a cytokine system such as the lymphokine, interleukin, monokine, chemokine, interferon or colony-stimulating factor system and/or a disturbance in the prostaglandin and/or leukotriene system. As used herein, the term xe2x80x9cpharmaceutical compositionxe2x80x9d comprises any composition suitable for human use as described in detail above.
The invention particularly relates to the use of IT9302 or a functional derivative thereof
for substantially inhibiting a biological effect in a human related to a cytokine for the prophylaxis or treatment of a condition related to a disturbance of a cytokine system; and/or
for substantially inhibiting a biological effect in a human related to a lymphokine for the prophylaxis or treatment of a condition related to a disturbance of a lymphokine system; and/or
for substantially inhibiting a biological effect in a human related to a interleukin for the prophylaxis or treatment of a condition related to a disturbance of a interleukin system; and/or
for substantially inhibiting a biological effect in a human related to a monokine for the prophylaxis or treatment of a condition related to a disturbance of a monokine system; and/or
for substantially inhibiting a biological effect in a human related to a chemokine for the prophylaxis or treatment of a condition related to a disturbance of a chemokine system; and/or
for substantially inhibiting a biological effect in a human related to a lymphokine for the prophylaxis or treatment of a condition related to a disturbance of a lymphokine system; and/or
for substantially inhibiting a biological effect in a human related to an interferon for the prophylaxis or treatment of a condition related to a disturbance of an interferon system; and/or
for substantially inhibiting a biological effect in a human related to a colony-stimulating factor for the prophylaxis or treatment of a condition related to a disturbance of a colony-stimulating factor system; and/or
for substantially inhibiting a biological effect in a human related to a prostaglandin for the prophylaxis or treatment of a condition related to a disturbance of a prostaglandin system; and/or
for substantially inhibiting a biological effect in a human related to a leukotriene for the prophylaxis or treatment of a condition related to a disturbance of a leukotriene system.